Week 8 pNIC-Bsa4 transformed with RpFabG RE Digest with SacII and PvuII (7/23/13)
Betty
pNIC RE Digest with RpFabG
elutions from miniprep of transformed pNIC
(with RpFabG)
Lane 1: empty
Lane 2: 1 kb DNA ladder
Lane 3: uncut pNIC-Bsa4 control
Lane 4: elution 1 (cut with SacII and PvuII)
Lane 5: elution 3 (cut with SacII and PvuII)
Lane 6: elution 4 (cut with SacII and PvuII)
Lane 7: pNIC (cut with SacII and PvuII)
Lanes 8-10: empty Week 7 pNIC-Bsa4 cut with BsaI-HF (7/19/13)
Betty
pNIC-Bsa4 cut with BsaI-HF
Lanes 1-3: empty
Lane 4: 1 kb DNA ladder (here, I was unsure if I had enough to show up, so I assumed it wouldn’t and added another ladder)
Lane 5: empty
Lane 6: 1 kb DNA ladder
Lane 7: cut pNIC-Bsa4 (cut with BsaI-HF)
Lane 8-10: empty
This was my third time making more cut plasmid; I made more in order to continue further rounds of cloning because none were, so far, working out.
RpFabG PCR squared product after PCR Cleanup (7/18/13)
The above two figures were my PCR squared product after nanodrop. They were made and cleaned 7/17, but nanodropped the morning of 7/18. The average concentration is 261.8 ng/uL.
Good job on these! - DR. B 071713 pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/17/13)
The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in five different tubes, each of volume of 50 uL as the protocol states. The five were then combined after the water bath and 15uL was taken to run a gel. The remaining 235uL were cleaned with the PCR cleanup kit and the results are shown above.
Week 6 pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13)
The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in four different tubes, each of volume of 50 uL as the protocol states. The four were then combined after the water bath and 20uL was taken to run a gel. The remaining 180uL were cleaned with the PCR cleanup kit and the results are shown above.
pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13) Figure 1. The first nanodrop of my cut pNIC-Bsa4 after PCR cleanup.
Figure 2. The second nanodrop of my cut pNIC-Bsa4 after PCR cleanup.
Overall, the concentration of my sample was low, but the 260/280 value of 1.8 represents the ideal purity in relation to the protein. The 260/230 value, however, was 1.1, lower than the desired 2.1 by nearly half.
pNIC-Bsa4 RE Digest (7/11/13)
Betty
RE Digest
Lane 1-2: empty
Lane 3: 1 kb DNA ladder
Lane 4: pNIC-Bsa4 (cut with BsaI-HF)
Lane 5-10: empty
I did a gel of pNIC-Bsa4 and cut it with BsaI to ensure that it would work. It cut twice, as it should, and the various sizes were appropriate.
pNIC-Bsa4 Midi Prep Nano Drop (7/10/13) Figure 1. The first nanodrop of my pNIC-Bsa4.
Figure 2. The second nanodrop of my pNIC-Bsa4.
I grew up pNIC-Bsa4, in 160mL LB Media with 160 uL of Kan, overnight on Monday and spun it down the day after. Today, I took the pellets and completed Midi Prep on it, the nanodropped the plasmid to determine the concentration. The average 260/280 value is 1.91 which is close to the desired 1.8, signifying a fairly high purity in relation to the protein. The 260/230 value, however, was 2.57, because it was fairly high in one nanodrop but a lot closer to 2.1 in the second.
PCR Squared (RpFabG Gene) Nano Drop after PCR Cleanup (7/09/13) Figure 1. The first nanodrop of the PCR cleanup results.
Figure 2. The second nanodrop of the PCR cleanup results.
The following are the nanodrop measurements for the PCR cleanup of Ricksettsia prowazekii FabG. The average concentration is 128.8 ng/uL, and the 260/280 average of 1.86 is very close to the desired 1.8 and the 260/230 average of 2.23 is very close to the desired 2.1. Week 5 Primary and Secondary PCR Gel (7/05/13)
Lane 1: empty
Lane 2: 1 kb DNA ladder
Lane 3: primary PCR
Lane 4: secondary PCR
Lane 5-10: empty
My primary PCR showed up like a smear as was desired, and my secondary PCR showed up at a band at between 100 and 200 base pairs.
PCR for pNIC-Bsa4 with pLIC (7/05/13)
Betty
pLIC for pNIC-Bsa4
Lane 1: empty
Lane 2: 100 bp DNA ladder
Lane 3: sample A
Lane 4: sample B
Lane 5: sample C
Lane 6: sample D (negative control)
Lanes 7-10: empty
While this PCR succeeded in that lane 5 (sample C) has the most DNA, my control evidently had some contamination.
PCR Green for pGFP (7/03/13)
Betty
Lane 1: 100 bp DNA ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)`
Lanes 6-10: empty
The GREEN PCR proved relatively successful, with equally sized bands and minimal contamination.
Virtual Plasmid for Rickettsia Prowazekii FabG in pNIC-Bsa4 (7/01/13) Forward Primer:
5’ TACTTCCAATCCATGATTGACCTCACGGGCAAAAC 3’ 35 bp GC Content 45.7% 0 mM Mg2+ Tm 64.4 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.1 oC 4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC
Reverse Primer:
5’ ACGGTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’
Reverse complement it:
5’ TATCCACCTTTACTGTTAAACCATCAGCATACCACCGT 3’ 38 bp GC Content 42.1% 0 mM Mg2+ Tm 64.1 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.2 oC 4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC
Week 4 Protein Purification NanoDrop (6/27/13) Figure 1. This nanodrop shows the results for Elution 1 of PfDXR.
Figure 2. This shows the results for Elution 2 of PfDXR.
RE Digest Gel (6/26/13)
BHLK REdigest pGBR22 62513
Lane 1, 5-7: Betty
Lane 8-10: Laraib
Lane 1: sample A-EcoRI (with NEBuffer 2)Lane 2: 1kb DNA ladder
Lane 3: uncut plasmid
Lane 4: skipped
Lane 5: sample B-PvuIILane 6: sample C-EcoRI+PvuII
Lane 7: sample D-EcoRI (with NEBuffer for EcoRI)
Lane 8: sample A-EcoRI (with NEBuffer 2)Lane 9: sample B-PvuII
Lane 10: sample C-EcoRI+PvuII
The RE Digest showed relatively good results, though was not completely correct.
Midi Prep DNA Sequencing Results (6/24/13)
I did Midi Prep on 6/19/13 and submitted a diluted 12 microliter sample of my pGBR22 plasmid to the DNA sequencing lab that same day. The sequencing was completed within two or three days, but I did not run the sequence through the BLAST database until Monday the 24th. The results were good, however: 100% Montipora efflorescens GFP-like chromoprotein.
Week 3 Agarose Gel (6/20/13)
BHLKpBGR2262013
Lanes 1-5: Betty
Lanes 6-10: Laraib
Lane 1: 100 bp ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 100 bp ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
The third time doing the pGBR22 PCR produced much better results than the previous two times.
DNA Sequencing (6/18/13)
I submitted my order of pGBR22 plasmid on 6/11/13 to the lab and received my results within a couple of days. However, I didn't check it against the BLAST database until today. The results showed that my sample was correctly matched at 100% to Montipora efflorescens GFP-like chromoprotein mRNA.
Agarose Gel (6/18/13)
BHLKVDS61813pGBR22
Lanes 1-5: Betty
Lanes 6-10: Laraib
Lane 1: 1 kb ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 1 kb ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
The bands showed up a lot better for my samples of pGBR22, but I proceeded to redo the PCR again anyway.
Week 2 - Betty great job. You will list your Weeks in reverse chronological order (you stared on Week 2 technically) . Thanks :) -- Dr. B Transformation Efficiency (6/10/13) Figure 1. Plate A, which contained 1ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate A showed no growth at all, demonstrating either an efficiency of zero or suggesting that something was done incorrectly or went wrong.
Figure 2. Plate B, which contained 5ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate B showed some growth, although only in a singular spot on the plate, suggesting that this plate was either done incorrectly or something else went wrong to show lack of growth.
something happened to the picture, so I have to reupload it from my email Figure 3. Place C had the most colonies. It contained 25ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates.
The numbers from the three plates were not similar, which could be expected due to the varying amounts of plasmid that were applied to each plate.
Agarose Gel of PCR Plasmid (6/13/13)
BHLKPCRpGBR22061313
First four lanes: Betty
Last four lanes: Laraib
Lane 1: 1 kb DNA ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 1 kb DNA ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
My four lanes showed no bars except for lane 3. In lane 5, no image would be expected, as it contained no DNA. However, lanes 2, 3, and 4 would have been expected to contain increasing amounts of visible bars, demonstrating amplification, as lane 2 contained the least amount of DNA and lane 4 contained the most.
pNIC-Bsa4 transformed with RpFabG RE Digest with SacII and PvuII (7/23/13)
Betty
pNIC RE Digest with RpFabG
elutions from miniprep of transformed pNIC
(with RpFabG)
Lane 1: empty
Lane 2: 1 kb DNA ladder
Lane 3: uncut pNIC-Bsa4 control
Lane 4: elution 1 (cut with SacII and PvuII)
Lane 5: elution 3 (cut with SacII and PvuII)
Lane 6: elution 4 (cut with SacII and PvuII)
Lane 7: pNIC (cut with SacII and PvuII)
Lanes 8-10: empty
Week 7
pNIC-Bsa4 cut with BsaI-HF (7/19/13)
Betty
pNIC-Bsa4 cut with BsaI-HF
Lanes 1-3: empty
Lane 4: 1 kb DNA ladder (here, I was unsure if I had enough to show up, so I assumed it wouldn’t and added another ladder)
Lane 5: empty
Lane 6: 1 kb DNA ladder
Lane 7: cut pNIC-Bsa4 (cut with BsaI-HF)
Lane 8-10: empty
This was my third time making more cut plasmid; I made more in order to continue further rounds of cloning because none were, so far, working out.
RpFabG PCR squared product after PCR Cleanup (7/18/13)
The above two figures were my PCR squared product after nanodrop. They were made and cleaned 7/17, but nanodropped the morning of 7/18. The average concentration is 261.8 ng/uL.
Good job on these! - DR. B 071713
pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/17/13)
The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in five different tubes, each of volume of 50 uL as the protocol states. The five were then combined after the water bath and 15uL was taken to run a gel. The remaining 235uL were cleaned with the PCR cleanup kit and the results are shown above.
Week 6
pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13)
The sample I nanodropped in figure 1 and 2 were created by cutting pNIC-Bsa4 in four different tubes, each of volume of 50 uL as the protocol states. The four were then combined after the water bath and 20uL was taken to run a gel. The remaining 180uL were cleaned with the PCR cleanup kit and the results are shown above.
pNIC-Bsa4 (cut with BsaI) Nano Drop after PCR Cleanup (7/12/13)
Figure 1. The first nanodrop of my cut pNIC-Bsa4 after PCR cleanup.
Figure 2. The second nanodrop of my cut pNIC-Bsa4 after PCR cleanup.
Overall, the concentration of my sample was low, but the 260/280 value of 1.8 represents the ideal purity in relation to the protein. The 260/230 value, however, was 1.1, lower than the desired 2.1 by nearly half.
pNIC-Bsa4 RE Digest (7/11/13)
Betty
RE Digest
Lane 1-2: empty
Lane 3: 1 kb DNA ladder
Lane 4: pNIC-Bsa4 (cut with BsaI-HF)
Lane 5-10: empty
I did a gel of pNIC-Bsa4 and cut it with BsaI to ensure that it would work. It cut twice, as it should, and the various sizes were appropriate.
pNIC-Bsa4 Midi Prep Nano Drop (7/10/13)
Figure 1. The first nanodrop of my pNIC-Bsa4.
Figure 2. The second nanodrop of my pNIC-Bsa4.
I grew up pNIC-Bsa4, in 160mL LB Media with 160 uL of Kan, overnight on Monday and spun it down the day after. Today, I took the pellets and completed Midi Prep on it, the nanodropped the plasmid to determine the concentration. The average 260/280 value is 1.91 which is close to the desired 1.8, signifying a fairly high purity in relation to the protein. The 260/230 value, however, was 2.57, because it was fairly high in one nanodrop but a lot closer to 2.1 in the second.
PCR Squared (RpFabG Gene) Nano Drop after PCR Cleanup (7/09/13)
Figure 1. The first nanodrop of the PCR cleanup results.
Figure 2. The second nanodrop of the PCR cleanup results.
The following are the nanodrop measurements for the PCR cleanup of Ricksettsia prowazekii FabG. The average concentration is 128.8 ng/uL, and the 260/280 average of 1.86 is very close to the desired 1.8 and the 260/230 average of 2.23 is very close to the desired 2.1.
Week 5
Primary and Secondary PCR Gel (7/05/13)
Lane 1: empty
Lane 2: 1 kb DNA ladder
Lane 3: primary PCR
Lane 4: secondary PCR
Lane 5-10: empty
My primary PCR showed up like a smear as was desired, and my secondary PCR showed up at a band at between 100 and 200 base pairs.
PCR for pNIC-Bsa4 with pLIC (7/05/13)
Betty
pLIC for pNIC-Bsa4
Lane 1: empty
Lane 2: 100 bp DNA ladder
Lane 3: sample A
Lane 4: sample B
Lane 5: sample C
Lane 6: sample D (negative control)
Lanes 7-10: empty
While this PCR succeeded in that lane 5 (sample C) has the most DNA, my control evidently had some contamination.
PCR Green for pGFP (7/03/13)
Betty
Lane 1: 100 bp DNA ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)`
Lanes 6-10: empty
The GREEN PCR proved relatively successful, with equally sized bands and minimal contamination.
Virtual Plasmid for Rickettsia Prowazekii FabG in pNIC-Bsa4 (7/01/13)
Forward Primer:
5’ TACTTCCAATCCATGATTGACCTCACGGGCAAAAC 3’ 35 bp
GC Content 45.7%
0 mM Mg2+ Tm 64.4 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.1 oC
4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC
Reverse Primer:
5’ ACGGTGGTATGCTGATGGTTTAACAGTAAAGGTGGATA 3’
Reverse complement it:
5’ TATCCACCTTTACTGTTAAACCATCAGCATACCACCGT 3’ 38 bp
GC Content 42.1%
0 mM Mg2+ Tm 64.1 oC 1.5 mM Mg2+ Tm 71.7 oC 2 mM Mg2+ Tm 72.2 oC
4 mM Mg2+ Tm 73.1 oC 6 mM Mg2+ Tm 73.5 oC
Week 4
Protein Purification NanoDrop (6/27/13)
Figure 1. This nanodrop shows the results for Elution 1 of PfDXR.
Figure 2. This shows the results for Elution 2 of PfDXR.
RE Digest Gel (6/26/13)
BHLK REdigest pGBR22 62513
Lane 1, 5-7: Betty
Lane 8-10: Laraib
Lane 1: sample A-EcoRI (with NEBuffer 2)Lane 2: 1kb DNA ladder
Lane 3: uncut plasmid
Lane 4: skipped
Lane 5: sample B-PvuIILane 6: sample C-EcoRI+PvuII
Lane 7: sample D-EcoRI (with NEBuffer for EcoRI)
Lane 8: sample A-EcoRI (with NEBuffer 2)Lane 9: sample B-PvuII
Lane 10: sample C-EcoRI+PvuII
The RE Digest showed relatively good results, though was not completely correct.
Midi Prep DNA Sequencing Results (6/24/13)
I did Midi Prep on 6/19/13 and submitted a diluted 12 microliter sample of my pGBR22 plasmid to the DNA sequencing lab that same day. The sequencing was completed within two or three days, but I did not run the sequence through the BLAST database until Monday the 24th. The results were good, however: 100% Montipora efflorescens GFP-like chromoprotein.
Week 3
Agarose Gel (6/20/13)
BHLKpBGR2262013
Lanes 1-5: Betty
Lanes 6-10: Laraib
Lane 1: 100 bp ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 100 bp ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
The third time doing the pGBR22 PCR produced much better results than the previous two times.
DNA Sequencing (6/18/13)
I submitted my order of pGBR22 plasmid on 6/11/13 to the lab and received my results within a couple of days. However, I didn't check it against the BLAST database until today. The results showed that my sample was correctly matched at 100% to Montipora efflorescens GFP-like chromoprotein mRNA.
Agarose Gel (6/18/13)
BHLKVDS61813pGBR22
Lanes 1-5: Betty
Lanes 6-10: Laraib
Lane 1: 1 kb ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 1 kb ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
The bands showed up a lot better for my samples of pGBR22, but I proceeded to redo the PCR again anyway.
Week 2 - Betty great job. You will list your Weeks in reverse chronological order (you stared on Week 2 technically) . Thanks :) -- Dr. B
Transformation Efficiency (6/10/13)
Figure 1. Plate A, which contained 1ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate A showed no growth at all, demonstrating either an efficiency of zero or suggesting that something was done incorrectly or went wrong.
Figure 2. Plate B, which contained 5ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates. Plate B showed some growth, although only in a singular spot on the plate, suggesting that this plate was either done incorrectly or something else went wrong to show lack of growth.
something happened to the picture, so I have to reupload it from my email
Figure 3. Place C had the most colonies. It contained 25ng of pGFP plasmid with DH5alpha bacteria on LB+Amp Agar plates.
The numbers from the three plates were not similar, which could be expected due to the varying amounts of plasmid that were applied to each plate.
Agarose Gel of PCR Plasmid (6/13/13)
BHLKPCRpGBR22061313
First four lanes: Betty
Last four lanes: Laraib
Lane 1: 1 kb DNA ladder
Lane 2: sample A
Lane 3: sample B
Lane 4: sample C
Lane 5: sample D (negative control)
Lane 6: 1 kb DNA ladder
Lane 7: sample A
Lane 8: sample B
Lane 9: sample C
Lane 10: sample D (negative control)
My four lanes showed no bars except for lane 3. In lane 5, no image would be expected, as it contained no DNA. However, lanes 2, 3, and 4 would have been expected to contain increasing amounts of visible bars, demonstrating amplification, as lane 2 contained the least amount of DNA and lane 4 contained the most.